Astrocytes, a major brain cell type, have key roles in brain function including extracellular ion buffering, de novo synthesis of neurotransmitters, transmitter uptake from the synaptic cleft, communication between the vasculature and neurons to regulate blood flow, modulation of neuronal activities, and responsiveness to injury. Astrocytes are highly coupled by gap junction channels, but the functional roles of the syncytium are not well understood because dye transfer studies cannot predict transfer of biological molecules and radiolabeled tracers can be metabolically transformed in donor and recipient cells. Our two initial findings, (1) very restricted transjunctional transfer of glucose-6-phosphate compared to other phosphorylated compounds related to the glycolytic pathway, and (2) marked impairment of dye transfer via astrocytic gap junctions by chronic hyperglycemia in vitro and in rat brain slices led to our hypothesis that transfer of molecules with fluxgenerating regulatory roles through astrocytic gap junctions is restricted, and hyperglycemia impairs gap junction-mediated shuttling of energy-related metabolites and signaling molecules within the astrocytic syncytium. The hypothesis is tested in three specific aims, (1) identify cytoplasmic molecules with fluxgenerating regulatory roles, redox signaling, or energy-donating or sensing roles that have restricted transjunctional transfer, (2) establish the role of connexin proteins in the specificity of transjunctional transfer of these biological molecules, and (3) establish the impact of chronic hyperglycemia on gap junction channelmediated trafficking. Our long-term goals are to understand the functions of the astrocytic syncytium, contributions of astrocytes to the brain[unreadable]s energy budget in health and disease, and the cellular basis of metabolic brain imaging. Novel methods are developed to assess selective permeability of connexin channels to biological molecules in cultured astrocytes, N2A cells stably transfected with different connexins, and adult rat brain slices. This proposal addresses some of the long-standing, basic issues related to connexin channel function [unreadable] permeability of biological molecules, functions of syncytial communication in situ, and development of assays, tools, or probes for studies of connexin structure and function. The translational component of the study focuses on impairment of gap junction trafficking during chronic hyperglycemia and development of a treatment protocol to restore the high glucose-induced deficit in gap junctional trafficking. The anticipated results will lead to a better understanding of nutritional sensing and coordination of energy production in gap junction-coupled astrocytes in normal and experimentally-diabetic rat brain and improve interpretation of brain imaging studies using technologies that detect signals arising from metabolic responses to changes in physiological activity